Composition comprising antigens and a mucosal adjuvant and a method for using

ABSTRACT

The majority of the mortality observed in young pigs occurs between three and five weeks post-weaning for  S. suis  infections and between four and six weeks post-weaning for  H. parasuis  and  Actinobacillus suis  infections. Clinical disease control associated with  S. suis, A. suis  and  H. parasuis  has been attempted using antibiotic treatment, by controlled exposure with live organisms, and by vaccination, using either inactivated commercial or autogenous bacterins administered parenterally. A similar lack of protection in very young pigs is observed with various viruses including swine influenza virus, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus and rotavirus. Disclosed herein is an immunogenic composition comprising inactivated antigens and a mucosal adjuvant. The composition may be administered to subjects, such as animals, particularly piglets from pre-weaning through the nursery phase, such as from birth or from three to five days of age, to protect from these diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/593,223, filed on May 11, 2017, which is a continuation-in-partapplication of International Application No. PCT/US2016/031902, filed onMay 11, 2016, and also claims priority to U.S. provisional patentapplication No. 62/334,971, filed on May 11, 2016. Both applications areincorporated herein by reference in their entireties.

FIELD

This disclosure relates to a composition comprising antigens and amucoadhesive adjuvant, and methods of making and using the composition.

BACKGROUND

Major economic losses in commercial swine nursery facilities are causedby diseases spread via the respiratory route. These includeStreptococcus suis-related infections, Actinobacillus suis-relatedinfections, Haemophilus parasuis related to Glässer's Disease,Mycoplasma infections caused by Mycoplasma hyorhinis or Mycoplasmahyosynoviae, Actinobacillus pleuropneumoniae-related infections,Pasteurella-related infections cause by Pasteurella multocida,infections caused by Bordetella bronchiseptica, Erysipelothrixrhusiopathiae, Salmonella spp. including Salmonella cholerasuis andSalmonella typhimurium, Escherichia coli, swine influenza viruses,porcine reproductive and respiratory syndrome viruses (PRRSv) anddisease caused by porcine epidemic diarrhea virus (PEDV). The majorityof the mortality observed in these young pigs occurs between three andfive weeks post-weaning for S. suis infections and between four and sixweeks post-weaning for H. parasuis infections. S. suis produces signsand/or symptoms including meningitis, polyserositis, arthritis,myocarditis, pericarditis and abortion. H. parasuis often produces anacute septicemia that leads to death. Additionally, the latter is animportant component in the Porcine Respiratory Disease Complex. A. suisand the Mycoplasma species are common respiratory diseases of pigs thatare transmitted by nose-to-nose contact. M. hyopneumoniae and M.hyorhinis both produce significant respiratory disease, whereas M.hyosynoviae more commonly causes arthritis resulting in lameness. B.bronchiseptica also results in a respiratory disease that is calledatrophic rhinitis. P. multocida is another organism associated withatrophic rhinitis and that is spread via mucous membranes. Swineinfluenza virus and PRRS also produce significant respiratory diseasesin pigs. PRRS is also associated with a reproductive syndrome causingabortion. On the other hand, the Salmonella species, E. coli and PEDVcause intestinal diseases that result in diarrhea that can kill neonatesand young very quickly.

Attempts have been made to control clinical disease associated with allof the bacterial species, including S. suis, H. parasuis and A. suis, byantibiotic treatment, by controlled exposure to live organisms, and byvaccination using inactivated, parenterally-injected bacterial vaccines(bacterins). A currently accepted strategy for protecting weaned pigsentering this stage of production involves vaccination using appropriatebacterins at the standard time of piglet processing followed byrevaccination at weaning. Such vaccinations utilize inactivated antigensthat are administered either intramuscularly or subcutaneously(parenteral).

In commercial settings, sows and gilts are often already exposed to thediseases for which their offspring, especially neonatal offspring, willbe susceptible. When sows or gilts are seropositive because of previousexposure or vaccination, they transfer lactogenic antibodies to theirneonatal offspring, which may protect the neonates for a period of time.Unfortunately, such maternal antibodies often interfere with the neonatebeing able to produce their own antibodies if they are vaccinated at tooyoung an age with a parenteral vaccine. Parenteral vaccination mainlystimulates the development of IgG and IgM antibodies.

In 1989, pork producers in the United States developed the Pork QualityAssurance program, a producer education and certification program toreduce the risk of animal health product residues in pork. In 2007, thisprogram was enhanced and became known as Pork Quality Assurance Plus(PQA)Plus®. One of the aspects of this program is to reduce the use ofparenteral injections, especially in young pigs, helping to ensure thesafety of food products.

SUMMARY

The present disclosure describes embodiments of a composition comprisinginactivated bacterial antigens and a mucosal adjuvant, such as amucoadhesive adjuvant. The composition can be administered mucosally,such as intranasally, to animals, such as pigs, particularly neonates,to immunize them again against typical neonatal and nursery diseases.Mucosal, such as intranasal, administration of the composition maystimulate the production of IgA, a mucosal antibody which may overcomematernal antibody interference. In a commercial setting, the intranasaladministration technique has several other distinct advantages whencompared with traditional parenteral vaccination. Such advantagesinclude ease of administration at processing, lessened risk of tissueloss due to needle use, and reduction of workload. Furthermore, themucosal route may be less susceptible to maternal immunity interference.

Mucosal vaccination, such as intranasal vaccination, is used fornon-inactivated antigens, such as administering a live virulent virus orbacteria, a live attenuated virus or bacteria, a modified live virus orbacteria, a live vectored viral or bacterial antigen, or a combinationthereof. However, those viruses or bacteria (herein defined as liveantigens) protect by causing either a low-level of infection or areplication in the animal, which stimulates an immune response. Noinactivated or killed antigens have been delivered mucosally, includingintranasally, to pigs and demonstrated to either stimulate an IgAresponse or produce a protective response. Such an intranasal method ofimmunizing pigs would also meet the requirements of the PQA.

Disclosed herein are embodiments of a composition comprising one or moreinactivated antigens obtained from one or more bacterial strains and/orone or more viral, strains, subunits, recombinant proteins and/orpeptides from bacterial or viral antigens, and a mucosal, such as amucoadhesive, adjuvant. Inactivated antigens that may be included insuch a composition are antigens from bacterial strains such as S. suis,H. parasuis, A. suis, M. hyorhinis, M. hyosynoviae, P. multocida, B.bronchiseptica, E. rhusiopathiae, S. cholerasuis, S. typhimurium, E.coli, C. perfringens, C. difficile, or combinations thereof; virusantigens that may be included in such compositions include but are notlimited to swine influenza viruses, such as H1N1, H3N2, H1N2, H2N3, or acombination thereof; porcine rotavirus groups A, B and C;

PRRS; and/or PEDV. In some embodiments, the composition comprises one ormore inactivated antigens obtained from one or more bacterial strains,such as S. suis, H. parasuis, and/or A. suis, and a mucosal, such as amucoadhesive, adjuvant. Other inactivated antigens that may be includedin such a composition are M. hyorhinis, M. hyosynoviae, P. multocida, B.bronchiseptica, E. rhusiopathiae, S. cholerasuis, S. typhimurium, E.coli, C. perfringens, C. difficile, swine influenza viruses, porcinerotavirus groups A, B and C, PRRS, and/or PEDV.

Other disclosed embodiments include certain inactivated viral antigenssuch as inactivated swine influenza (SIV or IAV-S), inactivated PorcineRespiratory and Reproductive Syndrome Virus (PRRSv), inactivated PorcineEpidemic Diarrhea Virus (PEDV) and other viruses that may be spread byinhalation of the virus.

In some embodiments, the composition does not comprise a nanoparticle.In other embodiments, the composition does not comprise a nanoparticlethat comprises one or more biodegradable polymers.

Also disclosed are embodiments of a method of administering thecomposition to a subject, particularly an animal, such as a pig. Thecomposition may be formulated for mucosal administration to pigs, suchas intranasal administration. Administration of the composition mayinduce an immune response in the subject. The immune response maycomprise an increased IgA response compared to an animal notadministered the composition. Mucosal administration may reduce theseverity and incidence of disease when the animal is later challenged byexposure to live organisms. Adjuvants with mucoadhesive characteristicsinclude, but are not limited to, polymers, such as those comprisingCarbopols or acrylic acids (such as polyacrylic acids), such asCarbigen™ adjuvant; oil-in-water based adjuvants, such as Emulsigen®adjuvant; nanoparticles; or combinations thereof. The adjuvants mayinclude immunostimulators, such as dimethyldioctadecyl ammonium bromide(DDA) or chloride (DDAC), pluronics, aluminum hydroxide, aluminumphosphate, and others known to persons of ordinary skill in the art.

Accordingly, disclosed embodiments concern an immunogenic compositioncomprising one or more strains of inactivated S. suis, one or morestrains of inactivated H. parasuis and/or one or more strains of A.suis. The disease-producing organisms including one or more isolates orstrains of H. parasuis, A. suis and/or S. suis may originally have beenisolated from a diseased animal. The bacteria can be grown in culturemedia to appropriate titers, such as at least 10⁵ colony formingunits/mL (CFU/mL), and then inactivated prior to incorporation into avaccine for administering mucosally.

Additionally, certain embodiments of the composition stimulate an IgAresponse when administered intranasally to neonatal pigs. Without beingbound to a particular theory, the IgA response may be able to overcomeinhibition by maternal antibodies that are delivered to the neonate bythe sow or gilt.

Certain disclosed embodiments include multivalent immunogeniccompositions comprising a combination of antigens from at least two ofS. suis, H. parasuis, and A. suis that have been inactivated with anacceptable inactivating agent and a mucosal, such as a mucoadhesive,adjuvant. The multivalent immunogenic composition may comprise antigensfrom all three organisms. The combination may be then administered topigs via a mucosal route. One or more administrations may be performedto produce protection from disease. Acceptable inactivating agents foruse with these antigens include, but are not limited to, formaldehyde,formalin, binary ethyleneimine, thimerosal, beta propiolactone,detergents such as NP40 and Triton X 100, and combinations thereof.Antigens may include whole culture bacteria, subunits that have beenextracted or separated from the culture, antigens obtained fromrecombinant organisms other than S. suis, H. parasuis or A. suis butwhich protect against S. suis, H. parasuis or A. suis infection orchallenge, or a combination thereof. Infection or challenge means thatthe animal suffers one or more clinical signs of the S. suis, A. suis orH. parasuis diseases when they have been exposed to these liveorganisms.

In specific embodiments, one or more of the S. suis, A. suis or H.parasuis antigens are present in the composition in an amount of fromabout 10² to about 10¹⁰ CFU/mL. The composition may further include asuitable pharmaceutical carrier, such as a diluent, adjuvant,antimicrobial agent, preservative, inactivating agent, or combinationthereof. Antimicrobial agents can include, but are not limited to,antibiotics such as gentamicin, penicillin, neomycin, polymyxin B andmycostatin.

Also disclosed herein are embodiments of a method for reducing theincidence or lessening the severity of clinical signs associated with orcaused by bacterial infections, such as infection by S. suis, A. suisand/or H. parasuis. In some embodiments, the method comprises reducingthe incidence or lessening the severity of clinical signs associatedwith or caused by additional bacterial infections, such as infection byM. hyorhinis, M. hyosynoviae, P. multocida, B. bronchiseptica, E.rhusiopathiae, S. cholerasuis, S. typhimurium, E. coli, swine influenzaviruses, PRRS, or PEDV comprising administering the composition to asubject, such as a pig, via a mucosal, such as intranasal, route. Andembodiments of a method of vaccinating swine against diseases of S.suis, H. parasuis, A. suis, M. hyorhinis, M. hyosynoviae, P. multocida,B. bronchiseptica, E. rhusiopathiae, S. cholerasuis, S. typhimurium, E.coli, swine influenza viruses, PRRS, PEDV, or a combination thereof, byadministering inactivated antigens of S. suis, H. parasuis, A. suis, M.hyorhinis, M. hyosynoviae, P. multocida, B. bronchiseptica, E.rhusiopathiae, S. cholerasuis, S. typhimurium, E. coli, swine influenzaviruses, PRRS, PEDV or a combination thereof, to a subject, such as apig, by an intranasal route, are also disclosed.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph of ELISA titer versus administration protocol,illustrating the measured IgA and IgG serum levels resulting from thevarious administration protocols, establishing that intranasaladministration resulted in the production of IgA and IgG antibodies inthe pigs.

FIG. 2 is a Western Blot of H. parasuis antigens, comparing pig antibodyresponses produced by intranasal (lanes 1, 2, and 3) or intramuscular(lanes 4, 5, and 6) vaccination with a combination of inactivated H.parasuis, A. suis, and S. suis, establishing that intranasaladministration resulted in protective antibody production in the pig.

FIG. 3 is a graph of Hemagglutination inhibition (HI) titer versustreatment group (TG), illustrating the HI titers by treatment group forH1N1 swine influenza virus in Example 6.

FIG. 4 is a graph of HI titer versus treatment group, illustrating theHI titers by treatment group for H3N2 swine influenza virus in Example6.

FIG. 5 is a graph of Hemagglutination inhibition (HI) titer versustreatment group (TG), illustrating the HI titers by treatment group forH1N1 swine influenza virus in Example 7.

FIG. 6 is a graph of HI titer versus treatment group, illustrating theHI titers by treatment group for H3N2 swine influenza virus in Example7.

FIG. 7 is a graph of IgA ELISA titers versus treatment group,illustrating the IgA titers for the various treatment groups in Example7.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R. §1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile, created on May 11, 2017, 20 MB, which is incorporated by referenceherein.

DETAILED DESCRIPTION I. Definitions

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. The singular forms“a,” “an,” and “the” refer to one or more than one, unless the contextclearly dictates otherwise. The term “or” refers to a single element ofstated alternative elements or a combination of two or more elements,unless the context clearly indicates otherwise. As used herein,“comprises” means “includes.” Thus, “comprising A or B,” means“including A, B, or A and B,” without excluding additional elements. Allreferences, including patents and patent applications cited herein, areincorporated by reference.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, percentages, temperatures, times, and soforth, as used in the specification or claims are to be understood asbeing modified by the term “about.”

Accordingly, unless otherwise indicated, implicitly or explicitly, thenumerical parameters set forth are approximations that may depend on thedesired properties sought and/or limits of detection under standard testconditions/methods. When directly and explicitly distinguishingembodiments from discussed prior art, the embodiment numbers are notapproximates unless the word “about” is recited.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting.

The term “adjuvant” as used herein means any component added to avaccine that enhances the immune response and includes any adjuvantsuitable for administration to an animal subject. In some embodiments,the adjuvant is, or comprises, a carbomer-based or carbopol-basedpolymer, such as Carbigen™ adjuvant (MVP adjuvants, a Division of PhibroAnimal Health, Omaha, Nebr., USA), HRA-3, and HRA-5; an aluminum salt,such as aluminum hydroxide or aluminum phosphate; a saponin such as QuilA® adjuvant or Stimulon™ adjuvant QS-21 (Antigenics, Framingham, Mass.);an oil-in-water adjuvant such as an Emulsigen® adjuvant (MVP adjuvants,a Division of Phibro Animal Health Corporation, Omaha, Nebr., USA)containing a non-metabolizable oil, paraffin oil, mineral and/orplant/vegetable and/or animal oils; a surfactant; a lipid; nanoparticlesincluding but not limited to oil-in-water based nanoparticles;cholesterol; water-in-oil emulsions; water-in-oil-in water emulsions;dimethyldioctadecyl ammonium bromide; dimethyldioctadecyl ammoniumchloride; or combinations thereof. In adjuvants comprising emulsions,the emulsion can be based in particular on light liquid paraffin oil,isoprenoid oil such as squalane or squalene, oil resulting from theoligomerization of alkenes, in particular of isobutene or decene, estersof acids or of alcohols containing a linear alkyl group, moreparticularly plant oils, ethyl oleate, propylene glycoldi-(caprylate/caprate), glyceryl tri-(caprylate/caprate) or propyleneglycol dioleate, esters of branched fatty acids or alcohols, inparticular isostearic acid esters. The oil may be used in combinationwith emulsifiers to form the emulsion. The emulsifiers are preferablynonionic surfactants, in particular esters of sorbitan, of mannide, ofglycol, of polyglycerol, of propylene glycol and of oleic, isostearic,ricinoleic or hydroxystearic acid, which are optionally ethoxylated, andpolyoxypropylene-polyoxyethylene copolymer blocks, in particular thePluronic products. In a preferred embodiment of the composition, theadjuvant is at a concentration of from 0.01% to 70%, such as from 1% to50%, 1% to 30%, 5% to 20%, 7% to 20%, or 10% to 20% by volume of thefinal product.

Bacterial antigens suitable for use in the present technology includeproteins, polysaccharides, lipopolysaccharides, and/or outer membranevesicles which may be purified, isolated or derived from a bacterium.Bacterial antigens also may include bacterial lysates and inactivatedbacteria formulations. In some embodiments, bacteria antigens may beproduced by recombinant expression. Typically, bacterial antigensinclude epitopes which are exposed on the surface of the bacteria duringat least one stage of a life cycle. Bacterial antigens may be conservedacross multiple serotypes. Bacterial antigens include antigens derivedfrom one or more of the bacteria disclosed herein.

Viral antigens suitable for use in the present technology includeinactivated (or killed) virus and/or viral proteins which may beisolated, purified or derived from a virus. Viral antigens can bederived from viruses propagated on a substrate, such as a cell cultureor other substrate, or they may be derived or expressed recombinantly.Typically, viral antigens include epitopes which are exposed on thesurface of the virus during at least one stage of a life cycle. Viralantigens may be conserved across multiple serotypes or isolates. Viralantigens include antigens derived from one or more of the virusesdisclosed herein.

The terms “mucoadhesive,” “mucosal adjuvant,” or “mucosally-adjuvanted”refer to an adjuvant that has the capability to adhere to mucosalmembranes and stimulate an immune response. Mucous membranes include thenasopharyngeal, oral, optic (eye), vaginal or anal membranes. The immuneresponse that is stimulated may include IgA, IgG, IgM, or a combinationthereof, which are found in the serum and in mucosal washings.Compositions comprising such adjuvants are applied to the mucosalmembranes of animals. Specific adjuvants with mucoadhesivecharacteristics include, but are not limited to, adjuvants comprisingpolymers, such as those comprising polyacrylic acids such as Carbopolsor Carbomers (e.g. Carbigen™ adjuvant, HRA-3, HRA-5, Carbigen-M,Carbigen-P, or combinations thereof); or oil-in-water based adjuvants,such as Emulsigen® adjuvant, Emulsigen®-D adjuvant, Emulsigen®-DL90adjuvant, Emulsigen®-BCL adjuvant, Emulsigen®P adjuvant, Emulsigen®-Madjuvant, and combinations thereof. Additionally, adjuvants containingnanoparticles can be used for intranasal administration. A person ofordinary skill in the art understands that a mucoadhesive adjuvant couldcontain any combination of the above adjuvants as well. Acceptablemucoadhesive adjuvants also include any adjuvant that when administeredmucosally, such as intranasally, stimulates an IgA response in pigsand/or protects pigs from challenge with a live organism.

Formulations for mucosal administration, including intranasalformulations, may comprise vehicles that neither cause irritation to thenasal mucosa nor significantly disturb ciliary function. Diluents suchas water, aqueous saline, phosphate buffered saline (PBS), culturemedias, or other known substances can be used in combination with othercomponents of disclosed compositions. The nasal formulations may alsocontain preservatives such as, but not limited to, gentamicin,formaldehyde, formalin, thimerosal, binaryethyleneimine, and/or betapropiolactone, and/or may contain neutralizing agents such as sodiumthiosulfate and/or sodium bisulfite. A surfactant may be present toenhance absorption of the subject proteins by the nasal mucosa.Acceptable surfactants include but are not limited to tweens, spans anddetergents such as NP40 and Triton X 100.

Acceptable inactivating agents for inactivating bacterial antigens, suchas S. suis, A. suis and/or H. parasuis antigens, or viral antigens suchas SIV (IAV-S), PRRS and PEDV, include, but are not limited to,formaldehyde, formalin, binary ethyleneimine, thimerosal,beta-propiolactone, and combinations thereof.

Infection or challenge means that the subject has been exposed to liveorganisms that may produce disease causing the subject to suffer one ormore clinical signs of the diseases when they have been exposed to theselive organisms.

The term “effective amount” or “therapeutically effective amount” refersto the amount of an active agent (such as one or more embodimentsprovided herein alone, in combination, or potentially in combinationwith other therapeutic agent(s)) sufficient to induce a desiredbiological result. That result may be amelioration or alleviation of thesigns, symptoms, or causes of a disease, or any other desired alterationof a biological system. The term “effective amount” or “therapeuticallyeffective amount” is used herein to denote any amount of a therapeuticand/or preventative that causes an improvement in a disease condition,or prevention of disease symptoms. The amount can vary with thecondition being treated, the stage of advancement of the condition, andthe type and concentration of formulation applied. Appropriate amountsin any given instance will be readily apparent to those of ordinaryskill in the art or capable of determination by routine experimentationsuch as vaccination and observation of an antibody response orvaccination followed by a challenge wherein the vaccinated animalsperform better than non-vaccinated animals that are challengedsimilarly.

II. Composition

Disclosed herein are embodiments of a composition comprising one or moreinactivated antigens and an adjuvant, such as a mucoadhesive adjuvant.The antigens may be any suitable antigen, but certain embodimentsparticularly may be obtained from one or more bacterial strains,particularly strains of S. suis, A. suis and/or H. parasuis and/or oneor more viruses such as SIV (IAV-S), PRRSv and PEDV. The composition maybe formulated for mucosal administration to a subject to stimulate animmune response, the response helping to reduce the severity andincidence of disease when the subject is later challenged by exposure tolive organisms. The subject may be a human or an animal. The term“animal” refers to a non-human animal. The animal may be a mammal, suchas a swine or pig. The immune response may comprise an IgA immuneresponse.

In some embodiments, the composition comprises inactivated antigens fromone or more S. suis strains or serovars, such as from 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more S. suis strains or serovars. In some embodiments,the composition comprises inactivated antigens from one or more H.parasuis strains or serovars, such as from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more H. parasuis strains or serovars. In some embodiments, thecomposition comprises inactivated antigens from one or more A. suisstrains or serovars, such as from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreA. suis strains or serovars. The composition may comprise inactivatedantigens from one or more S. suis strains or serovars and one or more H.parasuis strains or serovars; inactivated antigens from one or more A.suis strains or serovars and one or more H. parasuis strains orserovars; inactivated antigens from one or more S. suis strains orserovars and one or more A. suis strains or serovars, or inactivatedantigens from one or more S. suis strains or serovars, one or more H.parasuis strains or serovars, and one or more A. suis strains orserovars.

In some embodiments, the one or more antigens from S. suis comprise oneor more antigens from serovars ½, 2, or 3. Additionally, oralternatively, the composition may comprise one or more antigens havingat least a 85% sequence identity (i.e., 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%,99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) to any strain and/or serovarof S. suis currently known or later discovered or developed, such as,but not limited to, strains identified by the GenBank Accession numbersAM946016.1, FM252031.1, or NC_012926.1, and/or by SEQ ID NO: 1, SEQ IDNO: 2, or SEQ ID NO: 3, and particularly to serovars ½, 2, or 3. In someembodiments, the one or more antigens from H. parasuis comprise one ormore antigens from serovars 2 or 7. Additionally, or alternatively, thecomposition may comprise one or more antigens having at least a 85%sequence identity (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, 99.9% or 100%) to any strain and/or serovar of H. parasuiscurrently known or later discovered or developed, such as, but notlimited to, strains identified by the GenBank Accession numbersNZ_CP015099.1, NZ_CP009237.1, or NZ_CP009158.1, and/or by SEQ ID NO: 4,SEQ ID NO: 5, or SEQ ID NO: 6, and particularly serovars 2, or 7. Insome embodiments, the one or more antigens from A. suis comprise one ormore antigens having at least a 85% sequence identity (i.e., 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) to anystrain and/or serovar of A. suis currently known or later discovered ordeveloped, such as, but not limited to, the strain identified by theGenBank Accession number NZ_CP009159.1 and/or by SEQ ID NO: 7.

In some embodiments, the one or more antigens from SIV may comprise oneor more antigens having at least a 85% sequence identity (i.e., 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%)to any strain of SIV, such as, but not limited to, identified by GenBankAccession numbers M81707, KC676310.1, AF222769, JQ783083.1, KU942624.1,AF268128, AF268124, KP788773.1, KU229931, CY158137, and/or by SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. Insome embodiments, the one or more antigens from PEDV may comprise one ormore antigens having at least a 85% sequence identity (i.e., 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) to anystrain of PEDV, such as, but not limited to, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ IDNO: 24, or SEQ ID NO: 25. In some embodiments, the one or more antigensfrom PRRSV may comprise one or more antigens having at least a 85%sequence identity (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, 99.9% or 100%) to any strain of PRRSV, such as, but notlimited to, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,or SEQ ID NO: 30.

In some embodiments, the inactivated antigens include, but are notlimited to, whole culture bacteria; subunits obtained from the S. suis,A. suis and/or H. parasuis, and/or from any other bacteria or virus thatmay be included, that have been extracted or separated from the culture;subunits that have been extracted or separated from the cells; antigensobtained from recombinant organisms other than S. suis, A. suis or H.parasuis but which protect against S. suis, A. suis or H. parasuisinfection or challenge; or combinations thereof. Such antigens may alsobe combined with other antigens that are typically administered tosubjects, such as pigs, particularly neonatal pigs. Such additionalantigens include, but are not limited to, antigens from M. hyorhinis, M.hyosynoviae, P. multocida, B. bronchiseptica, E. rhusiopathiae, S.cholerasuis, S. typhimurium, E. coli, C. perfringens, C. difficile,porcine rotaviruses swine influenza virus (swine influenza virus, SIV),porcine reproductive and respiratory syndrome virus (PRRSv), porcineepidemic diarrhea virus (PEDv), porcine parvo virus, or a combinationthereof, and/or others known to persons of ordinary skill in the art. Insome embodiments, the inactivated antigens include, but are not limitedto, whole culture viruses; subunits or recombinants obtained from SIV(IAV-S), PRRSv, PEDV and/or from any other virus that may be included,that have been extracted or separated from the culture; subunits thathave been extracted or separated from the cells; antigens obtained fromrecombinant organisms other than SIV (IAV-S), PRRSv and PEDV but whichprotect against swine influenza, PRRSv or PEDV infection or challenge;or combinations thereof. Such antigens may also be combined with otherantigens that are typically administered to subjects, such as pigs,particularly neonatal pigs. Such additional antigens include, but arenot limited to, antigens from porcine rotaviruses, porcine parvo virus,or a combination thereof, and/or others known to persons of ordinaryskill in the art or any bacterial antigens such as M. hyorhinis, M.hyosynoviae, P. multocida, B. bronchiseptica, E. rhusiopathiae, S.cholerasuis, S. typhimurium, E. coli, C. perfringens, C. difficile, or acombination thereof.

In some embodiments, the composition does not comprise a nanoparticle.In some embodiments, the composition does not comprise a nanoparticlethat comprises one or more biodegradable polymers.

In some embodiments, administering the composition mucosally, such asintranasally, to a neonatal pig results in an IgA immune response thatalso may help to overcome maternal antibodies that the piglet mayreceive from a sow or gilt that is positive for S. suis, H. parasuis, A.suis, SIV (IAV-S), PRRSv, PEDV, rotavirus or any other bacteria orvirus. If the neonatal pig receives maternal antibodies and thenreceives a parenteral vaccination, the maternal antibodies often blockthe neonate from developing its own protective antibodies. The method ofmucosal vaccination, such as intranasal, administration described hereinmay produce an IgA response that can help overcome the maternalantibodies transferred to neonates.

Immunogenic compositions for mucosal administration, such as intranasaladministration, have several advantages over compositions that areadministered by other routes, such as the intramuscular or subcutaneousroutes. The advantages include, but are not limited to: 1) protectingthe neonatal pigs using humane techniques; 2) not exposing the neonateto stressful needle injections; 3) not involving injecting the neonateswith live or modified live organisms that can shed and spread disease;4) allowing the neonate to develop IgA antibodies that can overcomematernal IgG antibodies transferred from the sow or gilt; 5) not leavingany injection site lesions and thus allowing a zero day withdrawal time;6) being easier to administer and reducing the workload; 7) reducing orsubstantially eliminating the risk of accidental self-injection of theworker; and/or 8) being able to be administered in the face of anoutbreak to stop disease spread in a herd. Other advantages may beapparent from the description and the example section below.

Embodiments of the composition may be able to provide better protectionagainst diseases caused by S. suis, A. suis, H. parasuis, SIV (IAV-S),PRRSv, PEDV, rotavirus, and similar organisms that cause infectionsthrough the respiratory tract, following revaccination or additionaladministration of an immunogenic composition. The revaccination oradditional administration may be delivered via the intranasal route orby a parenteral route such as intramuscular, subcutaneous orintravenous. Furthermore, the disclosed composition may also be able toovercome maternal antibody inhibition of piglet antibody production,thus providing a greater protection of neonatal pigs throughout theirdevelopment, compared to neonatal pigs that are not administered thedisclosed composition.

Some embodiments of the disclosed composition are able to induceprotective levels of antibodies as measured by the ELISA in >50%,typically in >80% of the individuals that are administered thecomposition. In some embodiments, the mucoadhesive composition also isable to maintain protective levels of antibodies against the S. suis, A.suis, H. parasuis, SIV (IAV-S), PRRSv, PEDV and/or rotavirus strainsthroughout the piglet growth phase from the neonatal or pre-weaningphase through the nursery phase, which is typically from weaning atabout 21 days of age to about 10 weeks of age. Thus, in certainembodiments, the composition can produce a persistent immune responseagainst S. suis, A. suis, H. parasuis, SIV (IAV-S), PRRSv, PEDV and/orrelated diseases. As used herein, a “persistent immune response” refersto a protective antibody immune response which is capable of protectingthe pigs throughout their growth period in the nursery.

Embodiments of the immunogenic composition that comprise one or morestrains of inactivated S. suis, A. suis and/or inactivated H. parasuis,that are each independently grown to titers greater than 10² CFU/mL,preferably greater than 10⁵ CFU/mL, and more preferably greater than 10⁷CFU/mL, induce high titers of IgA antibodies in neonatal pigs. Such IgAtiters measured in serum will be at least 50 ELISA units, preferablygreater than 100 ELISA units.

Additional embodiments of the immunogenic composition that comprise oneor more strains of inactivated SIV (IAV-S), including but not limited toH1N1 and H3N2, PRRSv, PEDV, and/or inactivated rotavirus, that are eachindependently grown to titers greater than 10² TCID₅₀/mL, preferablygreater than 10⁵ TCID₅₀/mL, induce high titers of IgA antibodies inneonatal pigs. Such IgA titers measured in mucosal washings or in serumwill be at least 10 ELISA units, preferably greater than 50 ELISA units.

Surprisingly, embodiments of the disclosed immunogenic composition, whendelivered to neonatal pigs via the mucosal route, such as an intranasalroute, also induced serum IgG responses as demonstrated in the piglets(see FIGS. 1 and 2 and Table 2). In some embodiments, such titers arefrom 0 to 50 ELISA units, but in other embodiments, the titers aregreater than 50 ELISA units. It is expected that a mucosal IgA responsewill be measurable prior to the serum IgA response.

In some embodiments, the immunogenic composition comprises one or morestrains or isolates of inactivated S. suis, one or more strains orisolates of A. suis, one or more strains or isolates of H. parasuis, oneor more strains or subtypes of SIV (IAV-S), one or more isolates orgroups of PRRSv, one or more isolates of PEDV and/or one or moreisolates or types of rotaviruses which have been isolated from infectedpigs. In such embodiments, the strains may not have been passaged inartificial culture (in vitro) more than 20 times. Preferably, the one ormore strains of S. suis, A. suis, H. parasuis SIV (IAV-S), PRRSv, PEDVand/or rotavirus would not be passaged more than 10 times in vitro.

In some embodiments, the immunogenic composition is an inactivatedvaccine. The composition may be administered to a subject, such as apig, of any age. In some embodiments, the initial administration is topigs of from birth to 10 days of age, such as from one to ten days ofage, from one to five days of age, or from one to three days of age. Oneor more additional administrations of a composition comprising one ormore inactivated antigens obtained from one or more strains of S. suis,A. suis, H. parasuis, SIV (IAV-S), PRRSv, PEDV, and/or rotavirus may benecessary in two to six weeks from the initial administration of thedisclosed composition. This second administration may be deliveredparenterally and may include non-mucoadhesive adjuvants. Any additionaladministrations may be administered via a mucosal route, such asintranasal, or any other route that is preferred by the administrator.For instance, the additional administrations may be mucosal such asintranasal, intramuscular, subcutaneous, intraperitoneal, intravenous,or a combination thereof.

III. Examples Example 1—Composition Preparation

Two compositions were prepared for vaccination of neonates farrowed tosows in the farm described in Example 2. The object was to determinewhether intranasal administration of the disclosed composition toneonates within the first five days of life would help to reducemorbidity and mortality among the pigs. Multiple isolates of S. suis(serovars ½, 2, and 3) and H. parasuis (serovars 2, 7, and non-typeable)and one isolate of A. suis were identified and found to be responsiblefor causing clinical disease and mortality in the nursery pigs.Bacterins (inactivated bacterial vaccines) were prepared by growing allof the isolates in vitro to titers >10⁵ CFU/mL. Each isolate wasinactivated with 0.1% formalin after which isolates were pooled in equalamounts. After pooling, the pool was split into two equal aliquots. Onealiquot was first adjuvanted with 4% aluminum hydroxide. This wasfollowed by adding an oil-in-water adjuvant called Emulsigen® at 12%v/v. The second aliquot was adjuvanted with 10% Carbigen™, amucoadhesive polyacrylic acid adjuvant.

Example 2—Experimental Design

A commercial sow farm of 2,000 sows experiencing significant nurserymortality from S. suis, A. suis and/or H. parasuis infection wasselected for evaluation of the different administration routes/regimensof piglets. Nursery pigs from this source were also positive for porcinereproductive and respiratory syndrome virus (PRRS), porcine circovirustype 2 (PCV2), swine influenza A (IAV-S), and M. hyopneumoniae. Pigswere weaned at 21 days of age and transferred to a single-source,off-site nursery. The nursery contained four rooms which were filledwith approximately 900 pigs each. When clinical disease occurred in thenursery, individual animals were given an antibiotic injection. No feedor mass water medications were administered.

Neonatal piglets were enrolled at the sow farm into the study over fiveconsecutive weeks of farrowing. Administration occurred at the time ofprocessing which was between the third and fifth day post farrowing. Thepiglets were randomly assigned to one of four groups by colored eartags. The groups are shown in Table 1 and were:

1) intranasal (IN) administration at processing and repeated at weaning(about 21 days of age);

2) IN administration at processing and intramuscular (IM) injection atweaning;

3) IM administration at processing and repeated at weaning; and

4) IN administration at processing only (a single administration of thecomposition).

Piglets within litters were placed in different groups so as to equallyrepresent each of the four treatment groups as much as possible. Atweaning, treatment groups of pigs were not sorted to separate pens. Theanimals were randomly mixed together in order to promote uniformexposure to pathogens.

TABLE 1 Administration adjuvants and regimes Dose Age at Group Routes ofSize No. of No. of administration No. Adjuvant Administration (mL) DosesPiglets (Days) 1 Carbigen ™ IN/IN 2.0 2 911 3-5/21 2Carbigen ™/Emulsigen ® + IN/IM 1.0/2.0 2 911 3-5/21 Aluminum hydroxide 3Emulsigen ® + IM/IM 2.0/2.0 2 929 3-5/21 Aluminum hydroxide 4 Carbigen ™IN 1.0 1 848 3-5

Example 3—Laboratory Testing

From pigs in the week 3 production group, 10 serum samples were randomlycollected from each of the four treatment groups at approximately threeweeks after the weaning administrations. The samples were analyzed byhomologous ELISA using soluble proteins obtained from the H. parasuisvaccine strains as coated antigens in an assay method described by Lin,B. et al. “Antibody response of young pigs to autogeneous Haemophilusparasuis vaccine,” AASV 2007:207. IgA and IgG in serum samples weremeasured and are shown in Table 2 and FIG. 1.

The majority of animals in each of the treatment groups in thisinvestigation responded with measurable ELISA IgA titers to H. parasuis(vaccine strain) indicating that the pigs had largely (>80%)seroconverted. The IN/IM and IM/IM groups were the only treatments thatresulted in >80% rate of seroconversion as measured by IgG titer.Surprisingly, the IN groups developed significant IgG ELISA titers inthe serum.

TABLE 2 IgA and IgG Antibody responses post administration AntibodyResponse to H. parasuis as measured Group Routes of by ELISA No.Administration IgA IgG 1 IN/IN 320 60 2 IN/IM 220 540 3 IM/IM 580 560 4IN Single Dose 140 260

Example 4—Western Blot Analysis of Antibody Response to Antigens of H.Parasuis

Blood samples were obtained randomly from 10 pigs in each of the IN/INand IM/IM groups of pigs from Example 2. Equal aliquots were pooled andwere used in a Western Blot analysis to evaluate the antibody responsesof pigs to antigens of H. parasuis known to be important for protection.FIG. 2 provides the Western Blot for the IN/IN and IM/IM groups, withlanes 1, 2 and 3 representing the IN/IN group, and lanes 4, 5 and 6representing the IM/IM group. Lanes 1 and 4 are molecular weight markers(Bio-Rad #161-0373); lanes 2 and 5 are loaded with 20 ug/well of Hps#32575; and lanes 3 and 6 are loaded with 20 ug/well of Hps #31136.

The Western Blot analysis demonstrated that the IN/IN group of pigsdeveloped significant antibody responses to all of the importantprotective antigens of both H. parasuis strains that were included inthe inactivated vaccine that they received (FIG. 2). From top to bottom,the arrows in FIG. 2 indicate the presence of the bands at 82 kD(neurimidase), 48 kD (P2 adhesion protein) and the 35 kD outer membraneprotein, illustrating the presence of important antigens in the IN/INgroup. Surprisingly, the IN/IN group developed stronger antibodyresponses than the pigs in the IM/IM group.

Example 5—Results of Herd Administration

Over the five weeks of study observation, the parameters recorded werepre-weaning mortality, percent nursery mortality, percent treatedanimals (antibiotic injected upon presence of clinical disease), and thepercent poor quality pigs at the end of the nursery phase.

Data collected by treatment group for production week one through fourwere statistically analyzed. The week of production was used as theexperimental unit. Main effects ANOVA was performed with percent nurserymortality, total percent mortality (pre-weaning mortality plus nurserymortality), percent treated animals, and percent poor quality pigs asthe dependent variables and administration treatment as the independentvariable.

As noted in Table 3, during the nursery phase the IN/IN and IN/IM groupshad average mortality of 3.70% (s.d. 2.77%) and 9.13% (s.d. 6.67%),respectively. The average mortality observed in the IM/IM group was5.03% (s.d. 3.49%) whereas the average mortality observed in the INsingle dose (only once at processing) group was 1.42% (s.d. 1.79%).Statistical analysis indicated that across treatments, the type ofadministration did not significantly affect the mortality rate in thenursery (p>0.05). However, there is a strong indication that the singleintranasal vaccination produced the best result with the IN/INvaccination regimen producing the second best result.

TABLE 3 Average mortality during the nursery phase Routes of StandardGroup No. Administration Percent Mortality Deviation 1 IN/IN 3.70 2.77 2IN/IM 9.13 6.67 3 IM/IM 5.03 3.09 4 IN (single dose) 1.42 1.79

For total mortality (Table 4) which included both the pre-weaning andnursery mortality observed, the IN/IN and IN/IM groups had averagemortality of 12.7% (s.d. 2.61%) and 14.5% (s.d. 7.68%), respectively.The IM/IM group was 12% (s.d. 3.67%) whereas the average total mortalityobserved in the IN (single dose at processing age of 3-5 days) group was11.3% (s.d. 1.58%). Across treatments, a single dose of vaccineadministered intranasally at processing was as effective as two dosesadministered IN/IN, IN/IM or IM/IM (p>0.05). These results indicate thatthe greatest mortality occurred during the pre-weaning phase rather thanthe nursery phase.

TABLE 4 Average total mortality during both the pre-weaning and nurseryphases Routes of Standard Group No. Administration Percent MortalityDeviation 1 IN/IN 12.7 2.61 2 IN/IM 14.5 7.68 3 IM/IM 12.0 3.67 4 IN(single dose) 11.3 1.58

Table 5 shows the percentage of piglets treated with antibiotics. TheIN/IN and IN/IM groups had an average treatment mortality rate of 8.78%(s.d. 3.84%) and 9.53% (s.d. 6.17%), respectively. The IM/IM group was7.63% (s.d. 3.53%) while the average treatment rate observed in the IN(only at processing) group was 6.88% (s.d. 4.82%). Again, pigletsreceiving a single intranasal administration at 3-5 days of age orintranasal administration at 3-5 days of age followed by a secondintranasal administration at weaning (about 21 days) required no moreantibiotic treatments than piglets receiving regimens includingintranasal administration followed by intramuscular or two intramuscularadministration (p>0.05).

TABLE 5 Percentage of piglets treated with antibiotics during the studyRoutes of Standard Group No. Administration Percent Mortality Deviation1 IN/IN 8.78 3.84 2 IN/IM 9.53 6.17 3 IM/IM 7.83 3.53 4 IN (single dose)6.88 4.82

For nursery pigs grading as poor quality (see Table 6), the IN/IN andIN/IM groups had average poor quality of 22.60% (s.d. 4.91%) and 24.53%(s.d. 8.39%), respectively. “Poor quality” was defined as any pig thatcould be downgraded for any reason (for example, lighter weight). TheIM/IM group was 22.65% (s.d. 6.02%) whereas the rate of poor qualitypigs observed in the IN (only at processing) group was 22.23% (s.d.11.63%). Across treatments, the type of administration did notsignificantly affect the percentage of poor quality pigs observed at theend of the nursery phase (p>0.05).

TABLE 6 Percentage of poor quality piglets in the nursery Routes ofStandard Group No. Administration Percent Mortality Deviation 1 IN/IN22.60 4.91 2 IN/IM 24.53 8.39 3 IM/IM 22.65 6.02 4 IN (single dose)22.23 11.63

In summary, the nursery performance for the parameters measured,demonstrated that a single intranasal dose administered at 3-5 days ofage or two intranasal doses administered at 3-5 days of age and again atabout 21 days of age were as effective as regimens that includedintramuscular administration. However, the intranasal administration hasadditional advantages over intramuscular administration, including notinvolving stressful needle injections to neonatal pigs, not involvinginjecting the neonates with live or modified live organisms that canshed and spread disease, not leaving any injection site lesions and thusallowing a zero day withdrawal time, and being easier to administer andthus reducing the workload and worker's risk of accidentalself-injection for those administrating the composition. Furthermore,the intranasal administration may result in the neonate developing IgAantibodies that can overcome maternal IgG antibodies that aretransferred from the sow or gilt.

Example 6—Swine Influenza Virus Introduction

Swine influenza is an acute, highly contagious, respiratory diseaseresulting from type A swine influenza virus (SIV, also identified asIAV-S) infection. SIV is the leading cause of disease on swine farms inthe US, affecting 61.5% of wean-to-finish farms, 59.4% ofgrower/finisher farms, 46.2% of nursery farms, and 25.5% of sow farms.It has been estimated total economic damage of $1.3 billion between Mayand October of 2009 caused by the SIV epidemic in the US. Financiallosses attributable to SIV were estimated by one US swine producer at$10.31 per market pig. Vaccination is the main method of SIV preventionin pigs, with primary vaccination consisting of 2 injections 2 to 4weeks apart, with biannual booster vaccinations recommended for sows.Unlike for other swine diseases, such as leptospirosis andErysipelothrix, where vaccination levels on US breeding farms approach90%, typically only 46.4% of breeding farms vaccinate female pigs forSIV.

Adjuvants are substances that, when mixed with an antigen, enhance itsimmunogenicity. Adjuvants are often used to boost the immune responsewhen an antigen has low immunogenicity or when only small amounts of theantigen are available. For example, the antibody response of mice toimmunization with bovine serum albumin (BSA) can be increased fivefoldor more if the BSA is administered with an adjuvant. Adjuvants functionby prolonging antigen persistence, enhancing costimulatory signals,causing higher local inflammation, stimulating the nonspecificproliferation of lymphocytes, or by performing a combination of theabove. Good adjuvants can enable a reduction of the dose or theconcentration of the antigen within a vaccine, decreasing the cost ofthe vaccine. All data indicate that the selection of the appropriateadjuvant as well as the mass of antigen, both of which differ betweencommercial vaccines, are at least as important for the potency of SIVvaccines as the selection of the SIV strains in the vaccines.

Emulsion-based adjuvant systems have been widely utilized in vaccineformulation. Several different classes of emulsions exist, such asoil-in-water (O:W) emulsions, water-in-oil (W:O) emulsions,water-in-oil-in-water (W:O:W) emulsions, and protein-stabilizedemulsions. Water-in-oil-in-water emulsions contain water droplets withinlarger oil droplets, which are themselves suspended within a bulkaqueous solution. Whereas O:W emulsions are generally preferred forhuman applications, both W:O:W and W:O emulsions are widely used inveterinary vaccines. The advantages offered by W:O:W emulsions are theirlow viscosity as well as their ability to enhance the short and longterm immune responses. Oil-adjuvanted vaccines significantly increasehumoral immunity and generate higher antibody formation.

Typically, a hemagglutination inhibition (HI) titer of >1:40 isconsidered protective for SIV. However, it has been reported thatvaccination with an inactivated SIV vaccine in pigs does notconsistently generate complete immunity to virus challenges. The purposeof these experiments was to: 1) determine which MVP adjuvant wouldprovide the optimal serological response when using inactivated SIV asthe antigen; 2) evaluate whether a higher adjuvant concentration wouldproduce greater antibody responses to SIV; 3); evaluate theeffectiveness of a nanoparticle adjuvant delivered intramuscularly; and4) determine which MVP adjuvant could produce antibody responsesequivalent to or greater than an adjuvant included in one commerciallyavailable SIV vaccine for pigs.

Materials and Methods

Forty mixed sex commercial crossbreed swine of approximately 2 months ofage were used and identified with identical numbered ear tags in eachear. Pigs were allowed to acclimate to the research facility for 3 daysafter placement at the research facility prior to initiating the trial.Five pigs were randomly allocated to each of 8 treatment groups (TGs),as per the rubric in Table 7. FluSure XP SIV vaccine (Zoetis, Inc.;Serial number: A283483A/A280666; Expiration date: Dec. 3, 2013; Lotnumber: 1285595) antigen was used as the antigen for this experiment.This antigen is an inactivated SIV that is lyophilized and providedseparately from its commercial adjuvant.

TABLE 7 Experiment 1 Design TG Lot Number of Number¹ Adjuvant NumberPigs 1 Negative control; PBS 571 5 2 Commercially available SIV vaccine1285595 5 adjuvant 3 Emulsigen ®-D 20% formulation D1368 5 4Emulsigen ®-D 30% formulation D1368 5 5 Nanomune ™ 20% formulation062612 5 6 Nanomune ™ 30% formulation 062612 5 7 Emulsigen ®-DL90 20%formulation 062613 5 8 Emulsigen ®-DL90 30% formulation 062613 5 ¹TG =Treatment group; PBS = Phosphate buffered saline.

For all O:W emulsion adjuvants (Emulsigen®-D, Emulsigen®-DL90 andNanomune), the lyophilized antigen was reconstituted to the labeledvolume with sterile distilled water (DI) containing the concentration ofadjuvant noted in Table 7. For the commercially available inactivatedSIV vaccine, the adjuvanted diluent sold with the product was used toreconstitute the SIV antigen. Treatment Group 1 served as a negativecontrol group, and received an identical injection of phosphate bufferedsaline instead of receiving SIV antigen or adjuvant. A 2 mL dosecontaining vaccine plus adjuvant was injected into each pigintramuscularly in the right side of the neck. The adjuvant that each TGreceived was as follows: TG 1: PBS as negative control (Lot number:571); TG 2: adjuvant included in a commercially available SIV vaccine;TG 3: Emulsigen®-D formulated with antigen at a 20% concentration (MVPAdjuvants; Lot number: D1368); TG 4: Emulsigen®-D formulated withantigen at a 30% concentration (MVP Adjuvants; Lot number: D1368); TG 5:Nanomune, a nanoparticle adjuvant, formulated with antigen at a 20%concentration (MVP Adjuvants; Lot number: 062612); TG 6: Nanomuneformulated with antigen at a 30% concentration (MVP Adjuvants; Lotnumber: 062612); TG 7: Emulsigen®-DL90 formulated with antigen at a 20%concentration (MVP Adjuvants; Lot number: 062613); TG 8: Emulsigen®-DL90formulated with antigen at a 30% concentration (MVP Adjuvants; Lotnumber: 062613).

All pigs received 2 doses of vaccine and adjuvant based on theirrespective TGs; 1 dose on study day (D) 0, and 1 dose on D 21. Five to10 mL of venous blood was drawn from each pig at 3 time points; on D 0,prior to immunization; on D 21, prior to immunization, and on D 42.Blood samples from each time point were split into 2 equal aliquots. Onealiquot from each blood sampling day was used to test immunity using theHI assay by the Iowa State University (ISU) Veterinary DiagnosticLaboratory (VDL). Statistical analysis of serum antibody levels in thisexperiment was performed using repeated measures analysis of variance.

Results and Discussion

The results from this experiment are presented graphically in FIGS. 3and 4. On Day 0, prior to vaccination, several HI antibody titers werepositive, probably due to maternal antibody. Maternal SIV antibodylevels are dependent on antibody levels in the sow and decline inpiglets over a period of 4-14 weeks. It is noted that the TG1 titersdeclined from Day 0 to Day 42 so there was no SIV exposure during thestudy.

On D 21, using 1:40 as a cut-off point for successful generation ofprotective immunity to SIV, several pigs in the Emulsigen®-DL90 and theEmulsigen®-D treatment groups (TG 3, 4, 7, and 8) already possessedprotective HI antibody levels against SIV. On Day 21, for the H1N1strain of SIV, 20% of pigs in TG 3, 80% of pigs in TG 4, 80% of pigs inTG 7, and 60% of pigs in TG 8 possessed protective HI antibody levelswhereas 0% of the pigs in TG groups 1, 2, 5 and 6 demonstrated suchtiters. On Day 21, for the H3N2 strain of SIV, 20% of pigs in TG 3, 80%of pigs in TG 4, 20% of pigs in TG 7, and 60% of pigs in TG 8 possessedprotective HI antibody levels whereas 0% of the pigs in TG groups 1, 2,5 and 6 demonstrated protective HI antibody titers whereas. Earliergeneration of protective immunity ensures that pigs will be protected inthe event they face a disease challenge prior to receiving both doses ofvaccine. Additionally, these results indicate that even in the presenceof maternal antibody, Emulsigen®-D and Emulsigen®-DL90 could produceprotective titers at 21 days post a single vaccination.

Statistics

On Day 42, all pigs in all immunized treatment groups, TG 2-8, hadprotective levels of antibody for both the H1N1 and the H3N2 strains ofSIV. Emulsigen®-DL90 produced higher antibody titers than any otheradjuvant tested in this experiment, indicating the generation of thehighest level of protective immunity of all adjuvants tested in thisexperiment. Emulsigen®-D also produced very high antibody titers, morethan four-fold higher than the antibody titers produced by the adjuvantfrom a commercially available SIV vaccine (TG 2). Generation of higherantibody titer levels enables using lower levels of antigen, helping toreduce vaccine costs.

In this experiment, 3 of the analyzed adjuvants were tested at 2different formulation concentrations, 20% and 30%. For all 3 of theseadjuvants, the lower 20% concentration yielded higher antibody levelsthan did the 30% concentration. Adsorption of adjuvant to antigen isdetermined in part by the adjuvant:antigen ratio. Without being bound toa particular theory, an intermediate density of ligands, such asadjuvants, may provide statistically significant improvements in cellbinding in comparison with both higher and lower densities of adjuvants.Higher adjuvant densities may bind and mask too many antigen receptors,lowering the number of antigen receptors available to bind host cellsinvolved in the immune response and production of antibody, explainingthe lower antibody titers noted when using a higher concentration ofadjuvant.

Nanomune, a nanoparticle adjuvant, produced very low antibody titers.Adjuvants adsorb antigens and serve as a depot at the site of vaccineinjection, slowly releasing antigen into the body, thereby allowingantigen-specific lymphocytes to be exposed to antigen for a longerperiod of time. Nanoparticles, due to their small size, may diffuse fromthe site of injection more quickly, resulting in less exposure toantigen-specific lymphocytes, and hence generation of lower levels ofantibody. Further, adjuvant-antigen complexes involving nanoparticlesare smaller than adjuvant-antigen complexes involving larger-sizedadjuvants, lowering the likelihood of phagocytosis, in turn lowering theoverall immune response and lowering antibody level.

Example 7

This experiment was performed identically to the experiment described inExample 6, except that it utilized different TGs receiving differentadjuvants. Pigs were allocated to each of 8 TGs, as per the rubric inTable 8. Five pigs were allocated to TG 1-5 and TG 8. Ten pigs wereallocated to each of groups TG 6 and TG 7. Serum was collected from allpigs on D 0, D 21 and D42.

TABLE 8 Experiment 2 Design TG Lot Number Number¹ Adjuvant NumberAdministration² of Pigs 1 Negative control; 598 IM on D 0, D 21 5 PBS 2Commercially 1312332 IM on D 0, D 21 5 available SIV vaccine adjuvant 3Emulsigen ®- D1384 IM on D 0, D 21 5 D 20% 4 Emulsigen ®- 062014B IM onD 0, D 21 5 DL90 20% 5 Emulsigen ®- 17007 IM on D 0, D 21 5 BCL 20% 6Seppic W:O:W 50% 082814S IM on D 0, D 21 10* 7 MVP W:O:W 20% 082814M IMon D 0, D 21 10* 8 Carbigen ™ 10% 18053 Intranasally D 0, 5 D 21 ¹TG =Treatment group; PBS = Phosphate buffered saline; DDA =dimethyldioctadecylammonium bromide; W:O:W = Water-in-oil-in-waterimmersion; IM = intramuscular; D = Study day. ²All IM injectionsadministered in the right side of the neck; IM injections administeredas a 2 mL dose; Intranasal administration was 1 mL in each nostril, fora total dose of 2 mL. *10 pigs total were included in TG 6 and TG 7.

FluSure XP SIV vaccine (Zoetis, Inc.; Serial number: A309195/A310732;Expiration date: Mar. 3, 2015; Lot number: 1312332) an inactivated,lyophilized antigen was used as the antigen for this experiment. Thisantigen was mixed as follows with a different adjuvant for each TG. Forall O:W emulsion adjuvants (Emulsigen®-D, Emulsigen®-DL90 andEmulsigen®-BCL), the lyophilized antigen was reconstituted to thelabeled volume with sterile distilled water (DI) containing theconcentration of adjuvant noted in Table 8. For the commerciallyavailable SIV vaccine, the adjuvanted diluent sold with the antigen wasused to reconstitute the SIV antigen. For the Seppic W:O;W emulsion (ISA201) adjuvant, SIV antigen was reconstituted with 50% of the requireddiluent as PBS and the ISA 201 was added at a 50% concentrationaccording to directions provided by the manufacturer. For the MVP W:O:Wemulsion adjuvant, SIV antigen was reconstituted with 80% of therequired diluent after which the MVP W:O:W emulsion was added at a 20%concentration. For the Carbigen™ adjuvant, the adjuvant was first addedto the required volume of PBS, the pH was adjusted to between 6.0 and6.5 using 10N NaOH and then this was used to reconstitute thelyophilized SIV antigen to the concentration listed in Table 8.

Treatment Group 1 served as a negative control group, and received anidentical injection of PBS instead of receiving SIV antigen or adjuvant.The adjuvant that each TG received was as follows: TG 1: PBS as negativecontrol (Lot number: 598); TG 2: adjuvant included in a commerciallyavailable SIV vaccine; TG 3: Emulsigen®-D formulated at 20% with DDA(MVP Adjuvants; Lot number: D1384); TG 4: Emulsigen®-DL 90 formulated at20% with DDA (MVP Adjuvants; Lot number: 062014B); TG 5: Emulsigen®-BCLformulated at 20% (MVP Adjuvants; Lot number: 17007); TG 6: Seppic W:O:Wformulated at 50% (MVP Adjuvants; Lot number: 082814S); TG 7: MVP W:O:Wformulated at 20% (MVP Adjuvants; Lot number: 082814M); TG 8: Carbigen™formulated at 10% (MVP Adjuvants; Lot number: 18053).

A 2 mL dose containing vaccine plus adjuvant was injected into each pigintramuscularly in the right side of the neck for TGs 1-7. Treatmentgroup 8 received a 2 mL dose containing vaccine plus adjuvantadministered intranasally, with 1 mL administered into each nostril. Allpigs received 2 doses of vaccine and adjuvant; 1 dose on D 0, and 1 doseon D 21. Blood sampling and analysis was the same as described forexperiment 1 above. Statistical analysis of serum antibody levels inthis experiment was performed using repeated measures analysis ofvariance.

All serum samples were split into two aliquotes. One aliquot was sent toIowa State University diagnostic laboratory for testing ofhemagglutination inhibition titers. Samples were blinded as per groupand sampling day. Geometric mean data were analyzed statistically usingrepeated measures analysis of variance.

Results and Discussion

The results from this experiment are presented graphically in FIGS. 5-7,and in Tables 9 and 10.

TABLE 9 Hemagglutination Inhibition IgG Titers Day 0 Day 21 Day 42Adjuvant H1N1 H3N2 H1N1 H3N2 H1N1 H3N2 No Adj 0 0 0 0 2.5 12.5 Amphigen0 0 24 24 80 96 EM-D 20% 0 0 40 40 128 144 EM-DL90 20% 0 0 36 36 208 192EM-BCL 20% 0 0 22 22 112 96 SEPPIC WOW 20% 0 0 19 19 14 22 MVP WOW 20% 00 24 24 13 18 Carbigen 10% 0 0 8 0 0 12

TABLE 10 SIV Specific IgA response as measured by ELISA IgA Day 0 Day 42Control 0 0 Amphigen 0 0 EMULSIGEN-D 0 0 EMULSIGEN- 0 0 DL90 EMULSIGEN-0 0 BCL CARBIGEN-IN 0 400In this experiment, no positive antibody titers were found on Day 0,prior to immunization, indicating the lack of any maternal antibody,enabling definitive determination that all detected antibody resultedfrom immunization and not from any minor lingering levels of maternalantibody. On Day 21, for the H1N1 strain of SIV, 20% of pigs in TG 2,60% of pigs in TG 3, 80% of pigs in TG 4, 20% of pigs in TG 5, 10% ofpigs in TG 6, 20% of pigs in TG 7 and 0% of pigs in TG 8 possessedprotective HI antibody levels. On Day 21, for the H3N2 strain of SIV,20% of pigs in TG 3 possessed protective HI antibody levels. Moreimportantly, in this experiment, Emulsigen®-DL90 once again produced thehighest antibody levels followed by Emulsigen®-D, indicating thegeneration of the highest level of protective immunity of all adjuvantstested in this experiment. Generation of higher antibody titer levelsenables using lower levels of antigen, helping to reduce vaccine costs.

It was expected that the water-in-oil-in-water adjuvants (WOW) wouldproduce much higher antibody responses as this category of adjuvant isconsidered to be most effective with numerous antigens. Both WOWadjuvants produced extreme injection site reactivity as visuallyobserved by the attending veterinarian (data not shown) which wouldnormally suggest stimulation of a very high immune response.

The Carbigen™ adjuvant administered intranasally did not produce an IgGHI or ELISA response (data not shown). However, when IgA responses weremeasured using an ELISA specific for SIV IgA detection, this group (TG8) was the only group that produced a response which remained high at 42days (FIG. 7). Carbigen™ is an adjuvant that is known to havemucoadhesive properties. Therefore, it is more appropriate for use withintranasally administered antigens. In this case, an inactivatedantigen, when administered intranasally with Carbigen™ adjuvantstimulated a high IgA response as measured in the serum of the pigs.

CONCLUSION

In both Example 6 and Example 7, Emulsigen®-DL90 produced the highest HI(IgG) antibody levels, indicating the generation of the highest level ofprotective immunity of all adjuvants tested in these experiments.Emulsigen®-D also produced very high antibody titers. These titers weretwo to four fold higher than the antibody titers produced by theadjuvant from a commercially available SIV vaccine adjuvant. Both theseadjuvants produced higher titers than either a nanoparticle adjuvant ortwo WOW adjuvants evaluated in these Experiments. Additionally, it wasshown that a mucoadhesive adjuvant (Carbigen™) produced a very high IgAresponse in serum when used to adjuvant the inactivated SIV antigen.

Example 8 SIV (IAV-S) Early Proteins as Inactivated VaccinesAdministered Intranasally

In this example, antigens are prepared by extracting early proteins fromSIV (IAV-S) produced in tissue culture. Such proteins are called earlyproteins because they are produced within the first 2-10 hours of tissueculture infection by subtypes of SIV (IAV-S). Such subtypes aretypically H1N1 and H3N2 but can by any other of the subtypes of thisvirus. Early proteins are produced by infecting tissue culture cellsgrown in flasks, roller bottles or bioreactors with an SIV (IAV-S) virussubtype. Optionally, Trypsin can be used to enhance the infection withthe viruses. The infected cells are then incubated for between 2 and 10hours at 35-37° C. after which the supernatant is removed and the earlyproteins in the cells are released. The release of the early proteins isaccomplished by freezing and then thawing the cells sheet in thepresence of a buffer or by addition of a buffer such as one containingTRIS, EDTA and TRITON-X-100, NP40 or other equivalent surfactant.

The early proteins are then inactivated using any of the typicalinactivating agents such as binary ethyleneimine, formalin, formaldehydeor betapropiolactone. If a TRIS, EDTA, surfactant extraction process isused, an additional inactivating agent may not be necessary as the virusis already inactivated.

These early proteins of SIV (IAV-S) are then formulated into a vaccineby adding mucoadhesive adjuvant(s). One of these adjuvants can beCarbigen™ It is expected that this inactivated SIV (IAV-S) vaccine canbe administered intranasally to pigs to produce an IgA response thatwould be able to overcome the maternal antibody in such pigs. It is alsoexpected that such proteins may be cross-protective, unlike thehemagglutin and neuraminidase proteins of SIV (IAV-S).

IV. Statements

Disclosed here are embodiments of a composition formulated for mucosaladministration to an animal, the composition comprising inactivatedbacterial antigens and at least one mucoadhesive adjuvant. Themucoadhesive adjuvant may be selected for intranasal administration,and/or may comprise a polymer, an oil-in-water emulsion, a saponin, ananoparticle, a surfactant, a lipid, or a combination thereof. In someembodiments, the mucoadhesive adjuvant comprises a carbomer ornanoparticle, and may comprise polyacrylic acid.

In any of the above embodiments, the inactivated bacterial and/or viralantigens may be selected from antigens from Streptococcus suis,Haemophilus parasuis, Actinobacillus suis or a combination thereof, fromswine influenza virus (SIV or IAV-S), porcine reproductive andrespiratory syndrome virus (PRRSv), porcine epidemic diarrhea virus(PEDV), rotavirus, or a combination thereof, or any other types ofbacteria or viruses wherein disease is introduced via a mucosalmembrane. In certain embodiments, the inactivated bacterial antigenscomprise antigens from at least one strain or serovar of Streptococcussuis and at least one strain or serovar of Haemophilus parasuis;antigens from at least one strain or serovar of Haemophilus parasuis andat least one strain or serovar of Actinobacillus suis; and/or antigensfrom at least one strain or serovar of Streptococcus suis and at leastone strain or serovar of Actinobacillus suis. In particular embodiments,the inactivated bacterial antigens comprise antigens from at least onestrain or serovar of Streptococcus suis, at least one strain or serovarof Haemophilus parasuis and at least one strain or serovar ofActinobacillus suis.

In some embodiments, the inactivated antigens are selected from wholeculture bacteria, subunits that have been extracted or separated fromthe culture, extracts, antigens obtained from recombinant organismsother than S. suis, A. suis or H. parasuis but which protect against S.suis, A. suis or H. parasuis infection or challenge, inactivatedvector-delivered antigens, inactivated recombinant organisms carrying S.suis, A. suis, H. parasuis antigens, or a combination thereof.

The composition may further comprise one or more additional antigensselected from antigens of M. hyorhinis, M. hyosynoviae, P. multocida, B.bronchiseptica, E. rhusiopathiae, S. cholerasuis, S. typhimurium, C.perfringens, C. difficile, E. coli, porcine rotaviruses, swine influenzavirus, porcine reproductive and respiratory syndrome virus, porcineepidemic diarrhea virus, or porcine parvo virus.

The composition may further comprise an inactivating agent. In someembodiments, the inactivating agent is formaldehyde, formalin, binaryethyleneimine, thimerosal, beta propiolactone, or a combination thereof.

The composition may further comprise a diluent, preservative,antimicrobial agent, or a combination thereof.

In some embodiments, the inactivated antigens are present in an amountof from about 10² to about 10¹⁰ CFU/mL.

The animal may be a pig. In some embodiments, the animal is a neonatalor nursery pig.

In some embodiments, the composition is a vaccine.

In particular embodiments, the composition comprises inactivatedantigens of Streptococcus suis, Haemophilus parasuis, Actinobacillussuis or a combination thereof, and a mucoadhesive adjuvant comprisingpolyacrylic acid, and wherein the composition is formulated forintranasal administration.

Also disclosed herein is a method, comprising administering mucosally toan animal an effective amount of a first composition according to anyone of the embodiments disclosed herein. The animal may be a pig, and insome embodiments, the pig is a neonatal pig or a nursery pig. The pigmay be 10 days old or less, 5 days old or less, or 3 days old or less.

In some embodiments, administering mucosally comprises administratingintranasally.

Administering mucosally an effective amount of the first composition maycomprise administering mucosally an amount of the first compositionsufficient to produce an IgA immune response in the pig, compared to apig that is not mucosally administered the first composition.

In some embodiments, the method further comprises administering a secondcomposition comprising inactivated antigens of Streptococcus suis,Haemophilus parasuis, Actinobacillus suis or a combination thereof. Thesecond composition may be administered intranasally or intramuscularly.In some embodiments, administering the second composition comprisesadministering the second composition within two to six weeks of theadministration of the first composition.

Also disclosed herein are a method of vaccinating swine against diseasesof Streptococcus suis, Haemophilus parasuis, Actinobacillus suis or acombination thereof, the method comprising administering inactivated oneor more antigens from Streptococcus suis, Haemophilus parasuis,Actinobacillus suis or a combination thereof, to a pig by an intranasalroute. And a method for reducing the incidence or lessening the severityof at least one clinical sign associated with S. suis, A. suis or H.parasuis, the method comprising administering mucosally to a pig thecomposition according to any one of the above statements.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A composition formulated for mucosal administration to ananimal, the composition comprising inactivated bacterial antigens and atleast one mucoadhesive adjuvant, wherein the composition does notcomprise a nanoparticle.
 2. The composition of claim 1, wherein themucoadhesive adjuvant comprises polyacrylic acid.
 3. The composition ofclaim 1, wherein the inactivated bacterial antigens are selected fromantigens from Streptococcus suis, Haemophilus parasuis, Actinobacillussuis, swine influenza virus, porcine respiratory and reproductivesyndrome virus, porcine epidemic diarrhea virus, or a combinationthereof.
 4. The composition of claim 1, wherein the inactivatedbacterial antigens are selected from antigens from Streptococcus suis,Haemophilus parasuis, Actinobacillus suis, or a combination thereof. 5.A method, comprising intranasally administering to a swine, acomposition comprising an inactivated antigen and a mucoadhesiveadjuvant, wherein the composition does not comprise a nanoparticle.
 6. Amethod of inducing an immune response in a swine, comprisingadministrating to the swine the composition of claim
 1. 7. The method ofclaim 6, wherein inducing an immune response comprises inducing an IgAresponse in the swine.